Non-covalent dyes in microscale thermophoresis for studying RNA ligand interactions and modifications

Microscale Thermophoresis (MST) is a powerful biophysical technique that measures the mobility of biomolecules in response to a temperature gradient, making it useful for investigating the interactions between biological molecules. This study presents a novel methodology for studying RNA-containing samples using non-covalent nucleic acid-sensitive dyes in MST. This "mix-and-measure" protocol uses non-covalent dyes, such as those from the Syto or Sybr series, which lead to the statistical binding of one fluorophore per RNA oligo showing key advantages over traditional covalent labelling approaches. This new approach has been successfully used to study the binding of ligands to RNA molecules (e.g., SAM- and PreQ₁ riboswitches) and the identification of modifications (e.g., m⁶A) in short RNA oligos which can be written by the RNA methyltransferase METTL3/14.